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Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9.
The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.
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Genome editing in lysosomal disorders.
Lysosomal disorders are a group of heterogenous diseases caused by mutations in genes that encode for lysosomal proteins. With exception of some cases, these disorders still lack both knowledge of disease pathogenesis and specific therapies. In this sense, genome editing arises as a technique that allows both the creation of specific cell lines, animal models and gene therapy protocols for these disorders. Here we explain the main applications of genome editing for lysosomal diseases, with examples based on the literature. The ability to rewrite the genome will be of extreme importance to study and potentially treat these rare disorders.
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Stem cell-based and gene editing strategies in the treatment of congenital and acquired sensorineural hearing loss: a narrative review.
BACKGROUND: Sensorineural hearing loss (SNHL) represents the most common sensory deficit in humans and is increasingly prevalent with aging. Despite diverse etiologies, irreversible degeneration of cochlear hair cells and neurons remains a final pathway for auditory decline. OBJECTIVE: This narrative review synthesizes current preclinical and early clinical evidence on gene- and stem cell-based strategies for congenital and acquired SNHL, highlighting translational progress, delivery innovations, and persisting limitations. METHODS: A structured literature and registry search (PubMed, Embase, Scopus, Web of Science, ClinicalTrials.gov, EudraCT) was conducted using Boolean combinations of disease-, gene-, vector-, and delivery-related terms. From 140 initial records, 46 eligible studies were included after full-text screening. Data were qualitatively analyzed across two streams-human clinical (gene therapy) and preclinical/experimental (gene editing, RNA-based modulation, stem-cell regeneration). RESULTS: Two independent first-in-human AAV-mediated OTOF replacement trials in children with DFNB9 demonstrated partial restoration of hearing, establishing the first clinical proof-of-concept for cochlear gene therapy. Preclinical studies confirm durable auditory and vestibular rescue using dual-AAV systems, while CRISPR-based editing, antisense oligonucleotides (ASOs), and RNA interference (RNAi) approaches show mechanistic feasibility. Induced pluripotent stem cells (iPSCs) and inner-ear organoids replicate key developmental pathways, supporting modeling and future cellular repair. Delivery precision via the round window, oval window, and microneedle systems remains the major translational bottleneck. CONCLUSIONS: Gene- and cell-based approaches are transforming auditory regenerative medicine. Among them, AAV-mediated OTOF replacement has reached clinical validation, whereas CRISPR, ASO/RNAi, and iPSC-derived regeneration continue at advanced preclinical stages. Future success will depend on refining delivery, ensuring long-term safety, and harmonizing ethical and regulatory oversight.
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Applications of Genome Editing Technologies in CAD Research and Therapy with a Focus on Atherosclerosis.
Cardiovascular diseases, particularly coronary artery disease (CAD), remain the leading cause of death worldwide in recent years, with myocardial infarction (MI) being the most common form of CAD. Atherosclerosis has been highlighted as one of the drivers of CAD, and much research has been carried out to understand and treat this disease. However, there remains much to be better understood and developed in treating this disease. Genome editing technologies have been widely used to establish models of disease as well as to treat various genetic disorders at their root. In this review, we aim to highlight the various ways genome editing technologies can be applied to establish models of atherosclerosis, as well as their therapeutic roles in both atherosclerosis and the clinical implications of CAD.
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Generation of immunocompetent somatic glioblastoma mouse models through in situ transformation of subventricular zone neural stem cells.
Disease-relevant in vivo tumor models are essential tools for both discovery and translational research. Here, we describe a highly genetically tractable technique for generating immunocompetent somatic glioblastoma (GBM) mouse models using piggyBac transposition and CRISPR-Cas9-mediated gene editing in wild-type mice. We describe steps to deliver plasmids into subventricular zone endogenous neural stem cells by injection and electroporation, leading to the development of adult tumors that closely recapitulate the histopathological, molecular, and cellular features of human GBM. For complete details on the use and execution of this protocol, please refer to Garcia-Diaz et al.1.
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Prevention of acquired sensorineural hearing loss in mice by in vivo Htra2 gene editing.
BACKGROUND: Aging, noise, infection, and ototoxic drugs are the major causes of human acquired sensorineural hearing loss, but treatment options are limited. CRISPR/Cas9 technology has tremendous potential to become a new therapeutic modality for acquired non-inherited sensorineural hearing loss. Here, we develop CRISPR/Cas9 strategies to prevent aminoglycoside-induced deafness, a common type of acquired non-inherited sensorineural hearing loss, via disrupting the Htra2 gene in the inner ear which is involved in apoptosis but has not been investigated in cochlear hair cell protection. RESULTS: The results indicate that adeno-associated virus (AAV)-mediated delivery of CRISPR/SpCas9 system ameliorates neomycin-induced apoptosis, promotes hair cell survival, and significantly improves hearing function in neomycin-treated mice. The protective effect of the AAV-CRISPR/Cas9 system in vivo is sustained up to 8 weeks after neomycin exposure. For more efficient delivery of the whole CRISPR/Cas9 system, we also explore the AAV-CRISPR/SaCas9 system to prevent neomycin-induced deafness. The in vivo editing efficiency of the SaCas9 system is 1.73% on average. We observed significant improvement in auditory brainstem response thresholds in the injected ears compared with the non-injected ears. At 4 weeks after neomycin exposure, the protective effect of the AAV-CRISPR/SaCas9 system is still obvious, with the improvement in auditory brainstem response threshold up to 50 dB at 8 kHz. CONCLUSIONS: These findings demonstrate the safe and effective prevention of aminoglycoside-induced deafness via Htra2 gene editing and support further development of the CRISPR/Cas9 technology in the treatment of non-inherited hearing loss as well as other non-inherited diseases.
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Response to Levrier re: Human Germline and Heritable Genome Editing: The Global Policy Landscape.
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Transgene-free genome editing in citrus and poplar meristem tissues via biolistic ribonucleoprotein delivery of CRISPR-Cas9.
Biolistic particle bombardment was used to deliver CRISPR-Cas9 ribonucleoprotein complexes (RNP) into the shoot apical meristem tissue of citrus and axillary meristem tissue of poplar, generating directed mutations in target genes. The use of meristematic tissues offers a strategic approach to genome editing in woody species, especially those that are recalcitrant to conventional tissue culture, as these regions contain totipotent, highly regenerative cells capable of giving rise to whole plants. Here, we employed biolistic delivery of genome-editing reagents into theshoot apical meristem (SAM) of citrus and the axillary meristems (AXM) of poplar. The system was first validated using a GFP expression construct and subsequently applied for targeted genome editing. In citrus, edited plants were obtained at the CsNPR3 locus exclusively through the delivery of CRISPR/Cas9 ribonucleoproteins (RNPs), whereas plasmid-based vectors were unsuccessful. Similarly, genome editing in poplar was achieved using RNPs targeting the Pt4CL1 gene. Although chimeric events were detected, this approach provides a feasible and innovative framework for producing transgene-free edited perennial plants.
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In utero gene editing for monogenic lung disease.
Monogenic lung diseases that are caused by mutations in surfactant genes of the pulmonary epithelium are marked by perinatal lethal respiratory failure or chronic diffuse parenchymal lung disease with few therapeutic options. Using a CRISPR fluorescent reporter system, we demonstrate that precisely timed in utero intra-amniotic delivery of CRISPR-Cas9 gene editing reagents during fetal development results in targeted and specific gene editing in fetal lungs. Pulmonary epithelial cells are predominantly targeted in this approach, with alveolar type 1, alveolar type 2, and airway secretory cells exhibiting high and persistent gene editing. We then used this in utero technique to evaluate a therapeutic approach to reduce the severity of the lethal interstitial lung disease observed in a mouse model of the human SFTPCI73T mutation. Embryonic expression of SftpcI73T alleles is characterized by severe diffuse parenchymal lung damage and rapid demise of mutant mice at birth. After in utero CRISPR-Cas9-mediated inactivation of the mutant SftpcI73T gene, fetuses and postnatal mice showed improved lung morphology and increased survival. These proof-of-concept studies demonstrate that in utero gene editing is a promising approach for treatment and rescue of monogenic lung diseases that are lethal at birth.
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CRISPR/Cas9-Based Genome Editing: Understanding Differences in DNA Repair Pathways, Profiles, and Outcomes.
Over a decade of advances in Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-based technologies have culminated in the first-ever FDA-approved CRISPR/Cas-based therapy. Aside from this approved therapy for sickle cell anemia, several CRISPR/Cas-based therapies are currently under development or testing for a range of chronic diseases, including viral diseases like human immunodeficiency virus type 1 (HIV-1) infection, genetic diseases like familial hypercholesterolemia, and cancer. The success of these therapies hinges on the effective delivery of CRISPR/Cas9 components to target regions, efficient Cas endonuclease editing, repair profiles generated, and their resulting outcomes. Here, we discuss the factors that influence the generation of CRISPR/Cas9-generated repair edits, the overall profiles, and outcome prediction(s), as well as the analytical tools that have been developed to date. Finally, how this technology has been used towards a functional HIV-1 cure is discussed.
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